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FIBRILLAR ARRAYS OF A 110-kDa MYOSIN-LIKE PROTEIN INARABIDOPSISPISTILS
Cell Biology International, 1996, Vol. 20, No. 6, 451–453
SHORT COMMUNICATION
FIBRILLAR ARRAYS OF A 110-kDa MYOSIN-LIKE PROTEIN IN ARABIDOPSIS PISTILS JOHN W. LA CLAIRE II1* and RUSSELL H. GODDARD2 Department of Botany, University of Texas at Austin, Texas 78713 and 2Department of Biology, Valdosta State University, Valdosta, Georgia 31698, U.S.A.
1
Accepted 4 March 1996 K: Arabidopsis thaliana; cytoskeleton; immunofluorescence; myosin; pistil; plant cells.
INTRODUCTION The motor protein myosin has been implicated in many motile processes in plants (Nagai, 1993), and unravelling specific myosin functions awaits utilization of the molecular genetic approaches that have been successfully applied to studying myosin genes in protists and animals (Endow and Titus, 1992). Arabidopsis thaliana has become the most widely used angiosperm system for dissecting plant molecular genetics. Recently, myosin genes have been characterized from this model plant, and it appears that is has multiple myosin genes, some of which encode unconventional (i.e. small) myosins (e.g. Kinkema et al., 1994). Although there is a recent report on myosin localization in suspension culture cells of Arabidopsis (Yokota et al., 1995), the present work provides an initial investigation into whether its myosin exhibits a localized subcellular distribution in planta. MATERIALS AND METHODS Immunoblotting Arabidopsis thaliana tissues were powdered in a liquid nitrogen-cooled mortar, and lyophilized. Lyophilate (10 mg) was added to 200 ìl of 1# myosin extraction buffer (La Claire, 1991). The mixture was diluted 3:1 with 4# sample buffer, placed in a boiling water bath for 5 min, and *To whom correspondence should be addressed. 1065–6995/96/060451+02 $18.00/0
centrifuged at 13,000 rpm in a Sorvall RC-2B centrifuge (SH-MT rotor) for 10 min at 4)C. Supernatant was harvested and 75 ìl per lane was immediately subjected to polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes and subsequently handled exactly as reported previously (La Claire, 1991). Anti-myosin antibodies were affinity-purified from a rabbit antiserum raised against Dictyostelium myosin II HC (Berlot et al., 1985; La Claire, 1991). Secondary antibodies were alkaline phosphatase-labeled goat anti-rabbit immunoglobulin. Immunofluorescence Pistils from buds were excised and fixed in 4% paraformaldehyde in PNME (50 m PIPES-HCl, pH 6.9, 50 m NaCl, 1 m MgCl2, 1 m EGTA) for 30 min. After rinsing with 0.5# PNME, they were treated with 2% cellulase+0.5% pectinase (or macerase)+0.4 mannitol+1 m EGTA for 30– 120 min, and rinsed. Tissue fragments were submerged in a droplet of distilled water on a poly-Llysine-coated (5 mg/ml) slide, where they were gently teased apart. After rinsing in PBS (0.137 NaCl, 2.7 m KCl, 8.0 m KH2PO4, 1.5 m Na2HPO4, pH 7.3), the slides were air-dried. Cells were rehydrated with wetting solution (PBS containing 0.1–1.0% bovine serum albumin and 0.05% NaN3) for 30 min, and permeabilized in PBS with 2% Triton X-100 for 1 h. After rinsing, specimens were incubated for 3 h in wetting solution containing the affinity-purified anti-Dictyostelium myosin ? 1996 Academic Press Limited
452
Cell Biology International, Vol. 20, No. 6, 1996
II HC antibodies (50-fold dilution). Subsequent treatment with secondary antibodies and DAPI have been described (La Claire, 1991). 200
RESULTS 116
The affinity-purified antimyosin II HC antibodies appear to recognize a single band of c. 110 kDa in immunoblots of Arabidopsis protein extracts (Fig. 1). Stigma/style cells from floral buds display a distinctive and striking pattern of myosin immunofluorescence. Most cells have fibrous arrays of fluorescence, which may be straight or undulating in appearance (Fig. 2a–f). Many of the fibrils appear to radiate outward from the perinuclear region into the cytoplasm (Fig. 2a–f).
97
66
45
a
b
Fig. 1. Coomassie-blue stained electrophoresis gel of total soluble protein extract from Arabidopsis (a) with molecular weight marker positions indicated to the left in kDa. Antimyosin antibodies label a single band of c. 110 kDa (b).
DISCUSSION Very little is currently known about the distribution of myosin in higher plant cells, in general, primarily due to the lack of antibodies that will specifically recognize plant myosin. Although the
Fig. 2. Myosin immunofluorescence (a), DAPI staining (b) and Nomarski DIC (c) of a pair of pistil cells. A single myosin-containing fibril in the righthand cell partly surrounds the nucleus (arrow) and radiates into the cytoplasm. Myosin immunofluorescence (d) and DIC (e) show similar labeling patterns in a small clump of pistil cells. Multiple myosin-containing fibrils are also present in many cells (f). Scale bar represents 20 ìm.
Cell Biology International, Vol. 20, No. 6, 1996
data are severely limited at present, one generalization is that myosin is associated primarily with motility in higher plant cells (La Claire et al., 1995). Fibrillar arrays of myosin have not been reported previously in higher plant cells. We believe these fibers also contain actin, since perinuclear MF bundles have been observed in other floral cells of Arabidopsis (Webb and Gunning, 1994). Clearly, the structural and biochemical nature of these myosin-containing fibers must await ultrastructural and further immunological work. In a recent study of Arabidopsis cells grown in suspension culture, antibodies against a 170 kDa myosin from Lilium pollen revealed a punctate labeling pattern near the nucleus (Yokota et al., 1995). However, the antibodies used in the present study appear to be specifically recognizing fibrillar arrays of a small (unconventional) myosin-like protein given the 110 kDa size of the single band in immunoblots. Despite the fact that both myosins are partly associated with the perinuclear region, molecular genetic studies will be necessary to determine whether these two myosins have similar or unique functions. It would be interesting to determine if the Dictyostelium myosin antibodies recognize expression products of this or any of the other myosin genes that have been cloned from Arabidopsis. If not, our results suggest that these antibodies may be useful for isolating additional myosin genes from Arabidopsis expression libraries. ACKNOWLEDGEMENTS It is a pleasure of acknowledge Dr J. A. Spudich (Stanford University School of Medicine, Palo
453
Alto, U.S.A.) for providing antibodies, and to Drs A. Lloyd, K. Sathasivan and Ms D. Sutton (University of Texas at Austin) for plant material. This work was funded in part by a grant from the National Science Foundation to J.W.L. REFERENCES B CH, S JA, D PN, 1985. Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium. Cell 43: 307–314. E SA, T MA, 1992. Genetic approaches to molecular motors. Ann Rev Cell Biol 8: 29–66. K M, W H, S J, 1994. Molecular analysis of the myosin gene family in Arabidopsis thaliana. Plant Mol Biol 26: 1139–1153. L C JW, II, 1991. Immunolocalization of myosin in intact and wounded cells of the green alga Ernodesmis verticillata (Kützing) Børgesen. Planta 184: 209–217. L C JW, II, C R, H DL, 1995. Identification of a myosin-like protein in Chlamydomonas reinhardtii (Chlorophyta). J Phycol 31: 302–306. N R, 1993. Regulation of intracellular movements in plant cells by environmental stimuli. Int Rev Cytol 145: 251–310. W MC, G BES, 1994. Embryo sac development in Arabidopsis thaliana. II. The cytoskeleton during megagametogenesis. Sex Plant Reprod 7: 153–163. Y E, MD AR, L B, S T, P BA, 1995. Localization of a 170 kDa myosin heavy chain in plant cells. Protoplasma 185: 178–187.
SHORT COMMUNICATION
FIBRILLAR ARRAYS OF A 110-kDa MYOSIN-LIKE PROTEIN IN ARABIDOPSIS PISTILS JOHN W. LA CLAIRE II1* and RUSSELL H. GODDARD2 Department of Botany, University of Texas at Austin, Texas 78713 and 2Department of Biology, Valdosta State University, Valdosta, Georgia 31698, U.S.A.
1
Accepted 4 March 1996 K: Arabidopsis thaliana; cytoskeleton; immunofluorescence; myosin; pistil; plant cells.
INTRODUCTION The motor protein myosin has been implicated in many motile processes in plants (Nagai, 1993), and unravelling specific myosin functions awaits utilization of the molecular genetic approaches that have been successfully applied to studying myosin genes in protists and animals (Endow and Titus, 1992). Arabidopsis thaliana has become the most widely used angiosperm system for dissecting plant molecular genetics. Recently, myosin genes have been characterized from this model plant, and it appears that is has multiple myosin genes, some of which encode unconventional (i.e. small) myosins (e.g. Kinkema et al., 1994). Although there is a recent report on myosin localization in suspension culture cells of Arabidopsis (Yokota et al., 1995), the present work provides an initial investigation into whether its myosin exhibits a localized subcellular distribution in planta. MATERIALS AND METHODS Immunoblotting Arabidopsis thaliana tissues were powdered in a liquid nitrogen-cooled mortar, and lyophilized. Lyophilate (10 mg) was added to 200 ìl of 1# myosin extraction buffer (La Claire, 1991). The mixture was diluted 3:1 with 4# sample buffer, placed in a boiling water bath for 5 min, and *To whom correspondence should be addressed. 1065–6995/96/060451+02 $18.00/0
centrifuged at 13,000 rpm in a Sorvall RC-2B centrifuge (SH-MT rotor) for 10 min at 4)C. Supernatant was harvested and 75 ìl per lane was immediately subjected to polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes and subsequently handled exactly as reported previously (La Claire, 1991). Anti-myosin antibodies were affinity-purified from a rabbit antiserum raised against Dictyostelium myosin II HC (Berlot et al., 1985; La Claire, 1991). Secondary antibodies were alkaline phosphatase-labeled goat anti-rabbit immunoglobulin. Immunofluorescence Pistils from buds were excised and fixed in 4% paraformaldehyde in PNME (50 m PIPES-HCl, pH 6.9, 50 m NaCl, 1 m MgCl2, 1 m EGTA) for 30 min. After rinsing with 0.5# PNME, they were treated with 2% cellulase+0.5% pectinase (or macerase)+0.4 mannitol+1 m EGTA for 30– 120 min, and rinsed. Tissue fragments were submerged in a droplet of distilled water on a poly-Llysine-coated (5 mg/ml) slide, where they were gently teased apart. After rinsing in PBS (0.137 NaCl, 2.7 m KCl, 8.0 m KH2PO4, 1.5 m Na2HPO4, pH 7.3), the slides were air-dried. Cells were rehydrated with wetting solution (PBS containing 0.1–1.0% bovine serum albumin and 0.05% NaN3) for 30 min, and permeabilized in PBS with 2% Triton X-100 for 1 h. After rinsing, specimens were incubated for 3 h in wetting solution containing the affinity-purified anti-Dictyostelium myosin ? 1996 Academic Press Limited
452
Cell Biology International, Vol. 20, No. 6, 1996
II HC antibodies (50-fold dilution). Subsequent treatment with secondary antibodies and DAPI have been described (La Claire, 1991). 200
RESULTS 116
The affinity-purified antimyosin II HC antibodies appear to recognize a single band of c. 110 kDa in immunoblots of Arabidopsis protein extracts (Fig. 1). Stigma/style cells from floral buds display a distinctive and striking pattern of myosin immunofluorescence. Most cells have fibrous arrays of fluorescence, which may be straight or undulating in appearance (Fig. 2a–f). Many of the fibrils appear to radiate outward from the perinuclear region into the cytoplasm (Fig. 2a–f).
97
66
45
a
b
Fig. 1. Coomassie-blue stained electrophoresis gel of total soluble protein extract from Arabidopsis (a) with molecular weight marker positions indicated to the left in kDa. Antimyosin antibodies label a single band of c. 110 kDa (b).
DISCUSSION Very little is currently known about the distribution of myosin in higher plant cells, in general, primarily due to the lack of antibodies that will specifically recognize plant myosin. Although the
Fig. 2. Myosin immunofluorescence (a), DAPI staining (b) and Nomarski DIC (c) of a pair of pistil cells. A single myosin-containing fibril in the righthand cell partly surrounds the nucleus (arrow) and radiates into the cytoplasm. Myosin immunofluorescence (d) and DIC (e) show similar labeling patterns in a small clump of pistil cells. Multiple myosin-containing fibrils are also present in many cells (f). Scale bar represents 20 ìm.
Cell Biology International, Vol. 20, No. 6, 1996
data are severely limited at present, one generalization is that myosin is associated primarily with motility in higher plant cells (La Claire et al., 1995). Fibrillar arrays of myosin have not been reported previously in higher plant cells. We believe these fibers also contain actin, since perinuclear MF bundles have been observed in other floral cells of Arabidopsis (Webb and Gunning, 1994). Clearly, the structural and biochemical nature of these myosin-containing fibers must await ultrastructural and further immunological work. In a recent study of Arabidopsis cells grown in suspension culture, antibodies against a 170 kDa myosin from Lilium pollen revealed a punctate labeling pattern near the nucleus (Yokota et al., 1995). However, the antibodies used in the present study appear to be specifically recognizing fibrillar arrays of a small (unconventional) myosin-like protein given the 110 kDa size of the single band in immunoblots. Despite the fact that both myosins are partly associated with the perinuclear region, molecular genetic studies will be necessary to determine whether these two myosins have similar or unique functions. It would be interesting to determine if the Dictyostelium myosin antibodies recognize expression products of this or any of the other myosin genes that have been cloned from Arabidopsis. If not, our results suggest that these antibodies may be useful for isolating additional myosin genes from Arabidopsis expression libraries. ACKNOWLEDGEMENTS It is a pleasure of acknowledge Dr J. A. Spudich (Stanford University School of Medicine, Palo
453
Alto, U.S.A.) for providing antibodies, and to Drs A. Lloyd, K. Sathasivan and Ms D. Sutton (University of Texas at Austin) for plant material. This work was funded in part by a grant from the National Science Foundation to J.W.L. REFERENCES B CH, S JA, D PN, 1985. Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium. Cell 43: 307–314. E SA, T MA, 1992. Genetic approaches to molecular motors. Ann Rev Cell Biol 8: 29–66. K M, W H, S J, 1994. Molecular analysis of the myosin gene family in Arabidopsis thaliana. Plant Mol Biol 26: 1139–1153. L C JW, II, 1991. Immunolocalization of myosin in intact and wounded cells of the green alga Ernodesmis verticillata (Kützing) Børgesen. Planta 184: 209–217. L C JW, II, C R, H DL, 1995. Identification of a myosin-like protein in Chlamydomonas reinhardtii (Chlorophyta). J Phycol 31: 302–306. N R, 1993. Regulation of intracellular movements in plant cells by environmental stimuli. Int Rev Cytol 145: 251–310. W MC, G BES, 1994. Embryo sac development in Arabidopsis thaliana. II. The cytoskeleton during megagametogenesis. Sex Plant Reprod 7: 153–163. Y E, MD AR, L B, S T, P BA, 1995. Localization of a 170 kDa myosin heavy chain in plant cells. Protoplasma 185: 178–187.